Synergy between Raf and BCL2 in abrogating the cytokine dependency of hematopoietic cells

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Abstract
The Raf oncoprotein plays critical roles in the transmission of mitogenic signals from cytokine receptors to the nucleus. There are three Raf family members: A-Raf, B-Raf and Raf-1. Conditionally active forms of the Raf proteins were created by ligating N-terminal truncated activated forms to the estrogen-receptor (ER) hormone-binding domain resulting in β-estradiol-inducible constructs. We introduced these chimeric ΔRaf:ER oncoproteins into the murine FDC-P1 hematopoietic cell line. Two different types of cells were recovered after drug selection in medium containing either cytokine or β-estradiol: (1) cytokine-dependent cells that expressed the ΔRaf:ER oncoproteins; and (2) Raf-responsive cells that grew in response to the ΔRaf:ER oncoprotein. Depending upon the particular ΔRaf:ER oncoprotein, cytokine-dependent cells were recovered 103 to 105 times more frequently than Raf-responsive cells. To determine whether BCL2 could synergize with the ΔRaf:ER oncoproteins and increase the frequency of cytokine-independent cells, cytokine-dependent ΔRaf:ER-expressing cells were infected with either a BCL2 containing retrovirus or an empty retroviral vector. BCL2 overexpression, by itself, did not relieve cytokine dependency of the parental cell line. However, BCL2 overexpression increased the frequency of Raf-responsive cells approximately five- to 100-fold. Cytokine-dependent ΔRaf:ER-infected cells entered the G1 phase of the cell cycle after cytokine withdrawal and entered S phase only after cytokine addition. Raf-responsive ΔRaf:ER cells entered the G1phase of the cell cycle after estrogen deprivation and re-entered the cell cycle after addition of either IL-3 or the estrogen receptor antagonist tamoxifen which activates the ΔRaf:ER constructs. Expression of the BCL2 oncoprotein often delayed the exit from the S and G2/M phases demonstrating the protective effects BCL2 provided to these Raf and BCL2 infected cells. The ΔRaf:ER cells expressed the ΔRaf:ER proteins and downstream MEK and ERK activities after β-estradiol treatment. Raf-responsive cells that were also infected with BCL2 expressed higher levels of BCL2 than the cells that were not infected with BCL2. Thus BCL2 can synergize with the activated Raf in the abrogation of cytokine dependency of certain hematopoietic cells. These cells will be useful in furthering our understanding of the roles of the Raf and BCL2 oncoproteins in hematopoietic cell growth, cell cycle progression and prevention of apoptosis.